Abstract:

The invention concerns methods for cell screening of agents capable of
modulating the activity of SCF.sup.Met30 complexes comprising the
following steps: (i) contacting the product to be tested with a modified
yeast strain, including (a) a hybrid sequence comprising a sequence
coding for a Met4 protein, in its wild or mutated form, fused in phase
with at least a sequence coding for an appropriate marker, said hybrid
sequence being expressed under the control of a promoter, active in the
yeast and optionally (b) a reporter transcriptional system, consisting of
a reporter gene placed under the control of an appropriate operating
sequence or an appropriate yeast promoter, (ii) adding methionine and
(iii) determining the level of expression and stability of the expressed
protein from the hybrid sequence, and their uses. The invention also
concerns plasmids and yeast strains capable of being used in said
methods.

Claims:

1. A method for cell screening of compounds capable of acting on
pathologies linked to the dysfunction of the ubiquitin-proteasome cascade
in humans, said method comprising,(i) bringing the product to be tested
into contact with a modified yeast strain containing (a) a hybrid
sequence comprising a sequence encoding a Met4 protein, in its wild-type
form or in a mutated form involved in the transcriptional activation of
the MET genes, fused in phase with at least one sequence encoding an
appropriate marker, said hybrid sequence being expressed under the
control of a promoter active in yeast and optionally (b) a reporter
transcriptional system consisting of a reporter gene placed under the
control of an appropriate operator sequence or of an appropriate yeast
promoter,(ii) adding methionine at repressive concentrations of between
0.03 mM and 20 mM, or at nonrepressive concentrations, and(iii)
determining the level of expression and stability of the protein
expressed from the hybrid sequence, either by visualization and/or
quantification, or by determination of the activity of the reporter gene.

2. The method as claimed in claim 1, comprising, in parallel,(iv) bringing
the product to be tested into contact with a modified yeast strain
containing a hybrid sequence comprising a sequence encoding a Met30
protein, in its wild-type form or in a mutated form involved in the
biosynthesis of sulphur amino acids, fused in phase with at least one
sequence encoding an appropriate marker, said hybrid sequence being
expressed under the control of a promoter active in yeast,(v) adding
methionine at repressive concentrations of between 0.03 mM and 20 mM, or
at nonrepressive concentrations, and(vi) determining the level of
expression and stability of the protein expressed from the hybrid
sequence, either by visualization and/or quantification, or by
determination of the activity of the reporter gene.

3. The method as claimed in claim 1, further comprising an additional
means using a cellular system of control, said means comprising,(vii)
bringing the product to be tested into contact with a modified yeast
strain containing a hybrid sequence comprising a sequence encoding a
Met28 protein, in its wild-type form or in a mutated form involved in the
transcriptional activation of the MET genes, fused in phase with at least
one sequence encoding an appropriate marker, said hybrid sequence being
expressed under the control of a promoter active in yeast,(viii) adding
methionine at repressive concentrations of between 0.03 mM and 20 mM, or
at nonrepressive concentrations, and(ix) determining the level of
expression and stability of the protein expressed from the hybrid
sequence, either by visualization and/or quantification, or by
determination of the activity of the reporter gene.

4. The method as claimed in claim 1, further comprising an additional
means using an acellular system of control which is based on measuring
the levels of transcription of the hybrid sequences and of the metabolic
genes MET16 and MET25, said means comprising(x) extracting the total RNAs
of the modified strains used either in (i), or in (iv), or in (vii)
and(xi) measuring the levels of transcription of the hybrid sequence and
that of the metabolic genes MET16 and MET25.

5. The method as claimed in claim 1, wherein the marker used for the
construction of the hybrid sequence is chosen from the group consisting
of: the antigenic peptides, the intrinsic fluorescence proteins, the
proteins with measurable enzymatic activity and the DNA-binding factors.

6. The method as claimed in claim 1, wherein the promoter allowing the
expression of the hybrid protein is chosen from the group consisting of
inducible promoters active in S. cerevisiae and constitutive promoters.

7. The method as claimed in claim 1, wherein the reporter gene present in
the transcriptional reporter system is chosen from the group consisting
of the reporter genes whose activity can be visualized by a calorimetric
method, and the metabolic genes, whose activity can be measured by a
growth test.

8. The method as claimed in claim 1, wherein said reporter gene is placed
under the control, either of the promoter of a MET gene, or of LexA
operators.

9. The method as claimed in claim 8, wherein said MET gene is MET3, MET10,
MET16, MET25 or MET28.

10. A method for cell screening of compounds capable of acting on the
pathologies linked to the dysfunction of the ubiquitin-proteasome cascade
in humans, said method comprising(xii) bringing the product to be tested
into contact with a modified yeast strain containing a reporter
transcriptional system consisting of a reporter gene placed under the
control of a promoter selected from the group consisting of inducible
promoters active in S. cerevisiae and constitutive promoters.(xiii)
adding methionine at repressive concentrations of between 0.03 mM and 20
mM, or at nonrepressive concentrations, and(xiv) comparing the activity
of the reporter gene in repressive conditions to the activity of the
reporter gene in nonrepressive condition.

11. The method as claimed in claim 10, wherein the promoter is activated
by the Met4 transcription factor.

12. The method as claimed in claim 10, wherein the reporter gene present
in the transcriptional reporter system is chosen from the group
consisting of the reporter genes whose activity can be visualized by a
calorimetric method, and the metabolic genes, whose activity can be
measured by a growth test.

13. The method as claimed in claim 10, wherein said reporter gene is
placed under the control, either of the promoter of a MET gene, or of
LexA operators.

14. The method as claimed in claim 13, wherein said MET gene is MET3,
MET10, MET16, MET25 or MET28.

15. A plasmid, comprising a hybrid sequence comprising a sequence encoding
a Met4 protein in its wild-type form or in a mutated form involved in the
transcriptional activation of the MET genes, fused in phase with at least
one sequence encoding an intrinsic fluorescence protein, it being
possible for said hybrid sequence to be expressed in yeast under the
control of a constitutive or inducible promoter.

16. The plasmid as claimed in claim 15, wherein said intrinsic
fluorescence protein is the GFP protein and said promoter is a
constitutive promoter selected from MET4, MET28 and MET30.

17. The plasmid as claimed in claim 15, wherein said intrinsic
fluorescence protein is the GFP protein and said promoter is the GAL1
inducible promoter.

18. A yeast strain stably modified with at least one plasmid as claimed in
claim 15.

19. A plasmid, comprising a hybrid sequence comprising a sequence encoding
a Met28 protein in its wild-type form or in a mutated form involved in
the transcriptional activation of the MET genes, fused in phase with at
least one sequence encoding an intrinsic fluorescence protein, it being
possible for said hybrid sequence to be expressed in yeast under the
control of a constitutive or inducible promoter.

20. The plasmid as claimed in claim 19, wherein said intrinsic
fluorescence protein is the GFP protein and said promoter is a
constitutive promoter selected from MET4, MET28 and MET30.

21. The plasmid as claimed in claim 19, wherein said intrinsic
fluorescence protein is the GFP protein and said promoter is the GAL1
inducible promoter.

22. A yeast strain stably modified with at least one plasmid as claimed in
claim 19.

23. A plasmid comprising a hybrid sequence comprising a sequence encoding
a Met30 protein in its wild-type form or in a mutated form involved in
the biosynthesis of sulphur amino acids, fused in phase with at least one
sequence encoding an intrinsic fluorescence protein, it being possible
for said hybrid sequence to be expressed in yeast under the control of a
constitutive or inducible promoter.

24. The plasmid as claimed in claim 23, wherein said intrinsic
fluorescence protein is the GFP protein fused with a peptide comprising 3
hemagglutinin antigenic units and said promoter is the GAL1 inducible
promoter.

25. The plasmid as claimed in claim 23, wherein said intrinsic
fluorescence protein is the GFP protein and said promoter is a
constitutive promoter selected from MET4, MET28 and MET30.

26. The plasmid as claimed in claim 23, wherein said intrinsic
fluorescence protein is the GFP protein and said promoter is the GAL1
inducible promoter.

27. A yeast strain stably modified with at least one plasmid as claimed in
claim 23.

Description:

[0001]The subject of the present invention is methods for cell screening
of compounds capable of modulating the activity of SCF ubiquitin-ligase
complexes and their uses.

[0002]The existence of the controlled degradation of proteins has been
known for more than thirty years, but the exact molecular mechanisms
involved in this process have only been described over the past few
years.

[0003]In eukaryotic cells, the main pathway for the selective degradation
of proteins outside the lysosomes involves a cascade of reactions which
lead, in a first instance, to the labeling of the proteins to be
destroyed with a polypeptide, consisting of 76 amino acids, called
ubiquitin. The addition of several ubiquitin molecules then targets the
protein thus modified toward the proteasome, where it is destroyed. This
is known as the ubiquitin-proteasome pathway.

[0004]The degradation of a protein being essentially an irreversible
process, the ubiquitin-proteasome system is recruited by the numerous
regulations and signaling pathways whose aim is to modify long term the
cellular processes, in particular the developmental pathways, the
regulations of the cell cycle and the responses to the presence of
pathogenic agents. It is clear that compatibility of such processes with
normal cell life requires tight control of the nature of the proteins to
be destroyed.

[0005]This necessary selectivity of the processes for degrading proteins
is achieved by the enzymes which catalyze the addition of the ubiquitin
molecules and which are known by the generic name of ubiquitin ligases.

[0006]The proteasome is a protein complex, composed of several subunits,
which recognizes proteins when they are modified by the attachment of
ubiquitin to their lysine residues. This ubiquitylation, prior to the
recognition of the target proteins by the proteasome, involves at least
three enzymatic complexes, called E1, E2 and E3. E1 catalyzes the
activation of ubiquitin by forming a thioester between itself and
ubiquitin, which is then transferred to the enzyme E2, a conjugating
enzyme. Finally, the E3 ubiquitin ligase facilitates the recognition of
the target by E2 or directly transfers the ubiquitin from E2 to the
substrate (Hochstrasser M. et al., (1996), Annu. Rev. Genet, 30,
405-439).

[0008]Whereas the factor E1 is common to all the degradation pathways and
only serves to activate ubiquitin, the selectivity of the complex
responsible for ubiquitylation is provided by E3 ubiquitin ligase, which
interacts both with E2 and with the substrate (Hershko A. et al., (1983),
J. Biol. Chem., 258, 8206-8214).

[0009]Two groups of ubiquitin ligases can be distinguished:

[0010]isolated E3 proteins, in particular the family of HECT ("Homologous
to E6-AP Carboxyl-Terminus") proteins which possess a carboxy-terminal
domain homologous to that of the human E6-AP protein, involved in the
formation of a catalytic intermediate with ubiquitin, and

[0012]Currently, although more than 15 proteins containing F-boxes have
been identified by means of their sequence homology in the yeast genome,
only three complexes have been described and characterized in this
organism, namely: SCF.sup.Cdc4, SCF.sup.Grr1 and SCF.sup.Met.sup.30. Each
of these complexes has as target one or more specific substrates which
will be degraded after ubiquitylation; thus SCF.sup.Cdc4 has as target
the CDK (cyclin-dependent kinase) inhibitors, Sic1p and Far1p,
SCF.sup.Grr1 has as target the G1 cyclins, Cln/Cln2, and SCF.sup.Met30
has as target the CDK inhibitor, Swep1 (Koepp D. M. et al., (1999),
reference cited). The inventors have shown that the SCF.sup.Met30 complex
also plays a role in the negative regulation of the metabolism of sulfur
amino acids (Thomas D. et al., (1995), Mol. Cell, Biol., 15, 6526-6534).

[0014]In humans, a complex homologous to the SCF.sup.Met30 complex, the
SCF.sup.β-TrCP complex, has also been identified. The β-TrCP
protein (β-transducing repeat containing protein) has been
described, for the first time, in the context of an infection with the
HIV-1 virus. It is an F-box protein, induced by the viral Vpu protein
(Margottin F. et al., (1998), Mol. Cell., 1, 565-574), which is involved
in the degradation of the CD4 cellular receptors present at the surface
of infected cells, and is necessary for obtaining infectious HIV virus
particles. Additional studies have made it possible to show that, in the
absence of infection with the HIV-1 virus, β-TrCP allows specific
recognition and the destruction of targets such as IκBα, an
inhibitor of the ubiquitous transcription factor NFκB, which
directly regulates the immune and inflammatory response, β-catenin,
a protein of the Wg/Wnt pathway, whose abnormal solubilization leads to
the activation of the transcription of oncogenic genes and which is
involved in several types of cancer (Hart M. et al., (1999), Curr. Biol.,
9, 207-210; Kroll et al., (1999), J. Biol. Chem., 274, 7941-7945).

[0016]The SCF complexes in fact constitute the prototypes of an even
larger superfamily of ubiquitin ligases, also comprising the APC
complexes (anaphase promoting complexes), which control cell division,
and the VCB (VHL-EloginC/ElonginB) complexes, in particular those which
are involved in certain rare forms of hereditary cancers and those which
contain an SOCS (suppressors of cytokine signaling) box.

[0017]In most of the signaling pathways which involve the proteasome, the
signaling cascade which follows is preserved:

[0018]A. activation or inhibition of a protein kinase, under the influence
of an extracellular stimulus,

[0020]C. recognition and ubiquitylation of the target protein
phosphorylated by the SCF complex, and

[0021]D. targeting of the ubiquitinylated protein toward the proteasome
where it is finally degraded. The dephosphorylated protein escapes
degradation and accumulates, including in the nucleus.

[0022]The characterization of the signaling pathways controlled by the SCF
complexes, in various eukaryotic organisms, shows their central role in
maintaining cellular homeostasis.

[0023]The inventors have thus identified, in yeast, the Met30 protein as a
factor involved in the transcriptional repression of the genes involved
in the biosynthesis of sulfur amino acids (cysteine, methionine and
S-adenosyl-methionine (AdoMet)) (Thomas D. et al., (1995), Mol. Cell.
Biol., 15, 6526-6534). in yeast, this metabolic pathway has a set of
about 25 genes (MET genes) most of which are strictly coregulated, that
is to say that in response to an intracellular increase in AdoMet, which
may be easily obtained by adding methionine to the yeast growth medium,
the transcription of these genes is blocked. In humans, the metabolism of
sulfur amino acids is very different because of the fact that, unlike
yeast, it is not capable of assimilating sulfate and its growth requires
a dietary supply of sulfur amino acids (methionine).

[0024]Previous studies have demonstrated the existence of at least five
different transcriptional factors in yeast which are necessary for the
transcriptional activation of the MET genes; among these factors, there
may be mentioned two leucine zipper proteins (bZIP), Met4 and Met28, two
zinc finger proteins, Met31 and Met32, and the Cbf1 protein which is also
a component of the yeast kinetochore (Thomas D. et al. (1997), Microbiol.
Mol. Biol. Rev., 61, 503-532). The Met4 factor in particular does not
have a known function homology in humans. Depending on the genes, various
combinations of factors combine into complexes which recognize specific
sequences upstream of the MET genes; thus the Cbf1-Met4-Met28 complex
attaches to the TCACGTC sequence upstream of the MET16 gene whereas the
Met4-Met28-Met31 and Met4-Met28-Met32 complexes recognize the AAACTGTG
motif upstream of the MET3 and MET28 genes (Kuras L. et al. (1997), EMBO
J., 16, 2441-2451; Blaiseau P. L. et al. (1998) EMBO J., 17, 6327-6336).
In all these complexes, the transcriptional activation of the various MET
genes depends only on a single activation domain carried by the Met4
subunit.

[0025]The dysfunction of the ubiquitin-proteasome pathway, a pathway for
the degradation of proteins, has been established in numerous pathologies
of extremely diverse natures, in particular cancers, genetic diseases,
Parkinson's disease, Alzheimer's disease, inflammatory syndromes and
viral infections. Thus, it is known that any mutation present on the
target proteins around phosphorylation sites abolishes recognition of the
mutated proteins by the SCF complexes and leads to their stabilization.
It was recently demonstrated that such mutations, which affect
β-catenin and prevent its destruction, are involved in tumor
transformation in numerous tissues (colon cancer, melanoma,
hepatocarcinoma and the like). By contrast, the excessive degradation of
β-catenin in the neurons has been implicated in Alzheimer's disease,
and is implicated in neuronal death by apoptosis which occurs in this
pathology.

[0027]Various methods for screening compounds which are active on the
ubiquitin cascade and the degradation of the proteasome have been
proposed and use a ubiquitin-ligase/specific substrate pair, preferably
of human origin, in which the substrate is a protein regulating a
cellular process whose excessive degradation or lack of degradation by
the ubiquitin-proteasome pathway is directly involved in the pathology to
be treated:

[0029]International application PCT WO 99/04033 and American patent U.S.
Pat. No. 5,932,425, for screening substances capable of treating
disorders characterized by an increase in the transcriptional activity of
the NF-κB factor (autoimmune diseases, inflammatory conditions,
cachexia, AIDS), describe a method which also uses a human
ubiquitin-ligase of the HECT family (RSC or KIAAN), capable of
specifically ubiquitylating the IκB protein which forms an inactive
cytoplasmic complex with the ubiquitous transcription factor NF-κB,
which regulates the immune and inflammatory response,

[0030]International application PCT WO 99/38969 describes a method which
uses an F-box protein (human β-TrCP protein) which binds to the Vpu
viral protein, in order to screen anti-HIV agents. Vpu which serves as
intermediate in the targeting of CD4 cell receptors toward the
ubiquitin-proteasome degradation pathway thus participates in the
reduction of the number of functional CD4+ T lymphocytes which is
responsible for the immunodeficiency linked to HIV infection.

[0031]In general, in these various methods, which use systems of human
origin, there is a direct link between the pathology to be treated and
the proteins the modulation of whose degradation is sought.

[0032]However, continuing their work, the inventors have shown that
unexpectedly the SCF.sup.Met30 complex controls the transcription of all
the genes of the sulfate assimilation pathway and that the
transcriptional repression of this pathway, in response to an increase in
the intracellular AdoMet concentration, results from the specific
degradation of Met4 which involves the SCF.sup.Met30 complex. The
existence of this control establishes for the first time the direct link
between the activity of the SCF.sup.Met30 complex and the regulation of
the metabolism of sulfur amino acids in yeast.

[0033]Taking advantage of the extreme conservation of the molecular
mechanisms involved in the ubiquitin-proteasome pathway for degrading
proteins in all eukaryotic cells, the inventors have developed a method
for the cellular screening of compounds capable of acting on the
pathologies linked to the dysfunction of the ubiquitin-proteasome cascade
in humans using this pathway in a manner which had never been envisaged.
They indeed selected a protein of the ubiquitin-proteasome pathway, whose
stability is not directly involved in the pathologies to be treated, but
which makes it possible to have very efficient screening tools which have
the advantage of being rapid and reliable.

[0034]The subject of the present invention is consequently methods for
cell screening of compounds or of agents capable of modulating the
activity of SCF.sup.Met30 complexes, characterized in that they comprise
the following steps:

[0035](i) bringing the product to be tested into contact with a modified
yeast strain containing (a) a hybrid sequence comprising a sequence
encoding a Met4 protein, in its wild-type or mutated form, fused in phase
with at least one sequence encoding an appropriate marker, said hybrid
sequence being expressed under the control of a promoter active in yeast
and optionally (b) a reporter transcriptional system consisting of a
reporter gene placed under the control of an appropriate operator
sequence or of an appropriate yeast promoter,

[0036](ii) adding methionine at repressive concentrations of between 0.03
mM and 20 mM, or at nonrepressive concentrations, and

[0037](iii) determining the level of expression and stability of the
protein expressed from the hybrid sequence, either by visualization
and/or quantification, or by determination of the activity of the
reporter gene.

[0038]In an advantageous embodiment of the methods according to the
invention, said methods may comprise the following steps in parallel:

[0039](iv) bringing the product to be tested into contact with a modified
yeast strain containing a hybrid sequence comprising a sequence encoding
a Met30 protein, in its wild-type or mutated form, fused in phase with at
least one sequence encoding an appropriate marker, said hybrid sequence
being expressed under the control of a promoter active in yeast,

[0040](v) adding methionine at repressive concentrations of between 0.03
mM and 20 mM, or at nonrepressive concentrations, and

[0041](vi) determining the level of expression and stability of the
protein expressed from the hybrid sequence, either by visualization
and/or quantification, or by determination of the activity of the
reporter gene.

[0042]In another advantageous embodiment of the methods according to the
invention, said methods may comprise, in addition, an additional means
using a cellular system of control, which will make it possible to ensure
the specificity of response of the hybrid systems or of the
transcriptional reporter systems used in steps (i) to (vi), said means
comprising the following steps:

[0043](vii) bringing the product to be tested into contact with a modified
yeast strain containing a hybrid sequence comprising a sequence encoding
a Met28 protein, in its wild-type or mutated form, fused in phase with at
least one sequence encoding an appropriate marker, said hybrid sequence
being expressed under the control of a promoter active in yeast,

[0044](viii) adding methionine at repressive concentrations of between
0.03 mM and 20 mM, or at nonrepressive concentrations, and

[0045](ix) determining the level of expression and stability of the
protein expressed from the hybrid sequence, either by visualization
and/or quantification, or by determination of the activity of the
reporter gene.

[0046]In another advantageous embodiment of the methods according to the
invention, said methods may comprise, in addition, an additional means
using an acellular system of control which is based on measuring the
levels of transcription of the hybrid sequences and of the metabolic
genes MET16 and MET25, said means comprising the following steps:

[0047](x) extracting the total RNAs of the modified strains used either in
step (i), or in step (iv), or in step (vii) and

[0048](xi) measuring the levels of transcription of the hybrid sequence
and that of the metabolic genes MET16 and MET25.

[0049]The subject of the present invention is also methods for cell
screening of compounds capable of modulating the activity of the
SCF.sup.Met30 complexes, characterized in that they comprise the
following steps:

[0050](xii) bringing the product to be tested into contact with a modified
yeast strain containing a reporter transcriptional system consisting of a
reporter gene placed under the control of an appropriate yeast promoter,

[0051](xiii) adding methionine at repressive concentrations of between
0.03 mM and 20 mM, or at nonrepressive concentrations, and

[0052](xiv) determining the activity of the reporter gene.

[0053]For the purposes of the present invention, mutated form of a protein
is understood to mean a protein modified either by insertion, deletion or
substitution of one or more amino acids.

[0054]For the purposes of the present invention, methionine is understood
to mean the (L) form or the (DL) form of methionine.

[0055]For the purposes of the present invention, the expression
appropriate operator sequence controlling the reporter gene is understood
to mean a sequence recognized by a DNA-binding factor fused in phase with
a Met4 protein, such as the LexA-Met4 fusion protein.

[0056]For the purposes of the present invention, the expression yeast
promoter controlling the reporter gene is understood to mean a promoter
activated by the Met4 transcription factor, such as a MET gene such as
MET3, MET10, MET14, MET16, MET25 or MET28.

[0057]In an advantageous embodiment of the methods according to the
invention, the marker used for the construction of the hybrid sequence is
chosen from the group consisting of: the antigenic peptides, for example
the hemagglutinin (Ha) antigenic unit, the intrinsic fluorescence
proteins, such as for example the green fluorescence protein (Green
Fluorescence Protein or GFP), the proteins with measurable enzymatic
activity, and the DNA-binding factors such as for example E. coli LexA.

[0058]In another advantageous embodiment of the methods according to the
invention, the promoter allowing the expression of the hybrid protein may
be either an inducible promoter active in S. cerevisiae, which may be
advantageously chosen from the group consisting of the promoter of the
GAL1 gene and the promoter of the CUP1 gene, or a constitutive promoter,
such as for example the promoter of the S. cerevisiae ADH1 gene.

[0059]In another advantageous embodiment of said methods, the reporter
gene is chosen from the group consisting of the reporter genes whose
activity can be visualized by a colorimetric method, such as for example
the P. putida XylE gene and the E. coli LacZ gene, and the metabolic
genes, whose activity can be measured by a growth test, such as the S.
cerevisiae HIS3, URA3, TRP1 and LEU2 genes.

[0060]Said metabolic genes may be alternatively and advantageously used as
genes for selecting modified yeasts containing a hybrid sequence and/or a
reporter transcriptional system, as defined above.

[0061]In another advantageous embodiment of said methods, said reporter
gene (present in the reporter transcriptional system) is placed under the
control:

[0062]either of the promoter of a MET gene, preferably MET3, MET10, MET14,
MET16, MET25 or MET28,

[0063]or of an operator sequence recognized by the LexA-Met4 fusion
protein (LexA operators).

[0064]In another advantageous embodiment of the methods according to the
invention, the yeast strains carry one or more mutations which increase
permeability to the products to be tested (Vidal M et al., (1999), Trends
in Biotechnol., 17, 374-381).

[0065]To carry out the methods according to the invention, it is possible
to use yeast strains possessing the genetic background of the W303 strain
of the S. cerevisiae yeast which is described by Bailis A. M. et al.,
(Genetics, (1990), 126, 535-547) or any other strain characterized by
said yeast.

[0066]The transformation of the yeast cells by exogenous DNA was carried
out using techniques known to persons skilled in the art, in particular
the technique described by Schiestl R. H. et al. (Curr. Genet., (1989),
16, 339-346), genetic techniques (sporulation, dissection and evaluation
of markers, and the like) are also known, and there may be cited in
particular those described by Sherman F. et al. (in Methods in Yeast
Genetics: a Laboratory Manual, (1979), Cold Spring Harbor, N.Y.) and the
reverse genetic techniques described by Rothstein R. (Methods in
Enzymology, (1991), 194, 281-301); as a technique for integration
directed at the locus by single homologous recombination (crossing over),
there may be mentioned that which is described in Orr-Weaver et al.
(1983; reference cited).

[0067]In accordance with the invention, the yeasts may be transformed with
plasmids constructed by conventional molecular biology techniques, in
particular according to the protocols described by Sambrook J. et al.
(Molecular cloning Laboratory Manual, 2nd edition, (1989), Cold Spring
Harbor, N.Y.) and Ausubel F. M. et al. (Current Protocols in Molecular
Biology, (1990-1999), John Wiley and Sons, Inc. New York).

[0068]In accordance with the invention, the activity of the reporter genes
and of the MET genes is measured, according to the reporter gene and the
promoter used, by techniques known per se, in particular calorimetric
techniques, enzymatic techniques, immunological techniques, fluorescence
techniques or techniques for selection on an appropriate growth medium.

[0069]In accordance with the invention, the activity of the SCR.sup.Met30
complex is determined by measuring the level of expression and of
stability of the protein encoded by the hybrid sequence and/or by the
activity of the transcriptional reporter system; indeed, the hybrid
protein encoded by said hybrid sequence advantageously preserves the
intrinsic properties of each of the two fused elements constituting it.
For example, the Met4 marker protein is selectively degraded (by addition
of methionine to the culture medium) by the ubiquitin-proteasome pathway
(property of Met4 protein) and is visualized, in accordance with the
associated marker: when the marker present in the hybrid protein is the
GFP protein, then the activity of the SCF.sup.Met30 complex is visualized
by observing and by quantifying the fluorescence of the GFP-Met4 or
GFP-Met30 hybrid protein; when the marker present in the hybrid protein
is the hemagglutinin (Ha) antigenic unit, then the activity of the
SCF.sup.Met30 complex is visualized by immunological techniques, such as
protein transfer techniques (Western Blotting), ELISA techniques or
immunoprecipitation techniques (Harlow E. et al., (1988), Antibodies, a
Laboratory Manual, Cold Spring Harbor, N.Y.); when the cells contain a
transcriptional reporter system containing the Xy1E gene, then the
activity of the SCF.sup.Met30 complex is visualized by vaporization, on
yeast cells, of catechol at a concentration of between 50 mM and 1 M, and
by measuring the appearance of a yellow color (Worsay M. J. et al.,
(1975), J. Bacteriol., 124, 7-13); when the cells contain a
transcriptional reporter system containing the E. coli LacZ gene, then
the activity of the SCF.sup.Met30 complex is visualized by measuring the
appearance of a blue color, on a medium containing the colorigenic
substrate X-Gal (Sambrook J. (1989), reference cited); when the cells
contain a transcriptional reporter system containing the S. cerevisiae
HIS3 gene, then the activity of the SCF.sup.Met30 complex is visualized
by growing the yeast on a minimum medium not containing histidine, in the
presence of aminotriazole, at concentrations of 0.5 mM to 200 mM.

[0070]Because of the existence of numerous subunits common to the various
SCF complexes, said subunits being in stoichiometric equilibrium, the
SCF.sup.Met30 complex may serve as a model for screening molecules for
their capacity to modulate either the activity of the signaling pathways
controlled by the SCF complexes as a whole, or that of the
ubiquitin-proteasome pathway, a pathway for the degradation of proteins.

[0071]Surprisingly, this second system (ubiquitin-proteasome pathway), as
used in the present invention, by using, as substrate, the Met4 protein,
is particularly advantageous for screening molecules or agents capable of
acting on pathologies linked to the dysfunction of the
ubiquitin-proteasome cascade in humans such as cancers, genetic diseases,
Parkinson's disease, Alzheimer's disease, inflammatory syndromes and
viral infections:

[0072]simplicity: the induction of the SCF.sup.Met.sup.30/Met4 complex
system is carried out simply by adding methionine to the growth medium,
and when the marker is the GFP protein, the activity of the SCF.sup.Met30
complex is visualized directly by observing and/or quantifying the
fluorescence emitted,

[0073]speed of development: the reverse genetic techniques, the integral
knowledge of the genome of the yeast and the molecular biology methods
adapted to this organism ensure a rapid use of indicator stable strains,

[0074]speed of growth and of screening: yeast is a rapidly growing and
high yield microorganism, which allows the production of modified cells
for a large number of screenings,

[0075]low cost: yeast is a microorganism whose culture, storage and
characterization are not very expensive.

[0076]The methods of screening according to the invention may serve in
particular to select active agents such as anticancer agents,
anti-inflammatory agents, antiviral agents or agents active in genetic
diseases, in particular in Parkinson's disease and Alzheimer's disease.

[0077]For the purposes of the present invention, compound or agent is
understood to mean any molecule derived from methods of syntheses or
natural resources.

[0078]The subject of the present invention is also the use of agents
selected by the methods according to the present invention, for the
preparation of medicaments intended for the treatment of diseases linked
to disorders of the activity of the SCF complexes or of the
ubiquitin-proteasome pathway, such as cancers, genetic diseases,
Parkinson's disease, Alzheimer's disease, inflammatory syndromes and
viral infections.

[0079]The subject of the present invention is also plasmids, characterized
in that they contain a hybrid sequence comprising a sequence encoding a
Met4, Met28 or Met30 protein in its wild-type or mutated form, fused in
phase with at least one sequence encoding a marker chosen from the Met
protein, from the group consisting of: antigenic peptides, intrinsic
fluorescence proteins and proteins with measurable enzymatic activity, it
being possible for said hybrid sequence to be expressed in yeast under
the control of a constitutive or inducible promoter.

[0080]In accordance with the invention:

[0081]when said plasmid comprises a sequence encoding a Met4 protein, then
said marker is chosen from antigenic peptides, intrinsic fluorescence
proteins,

[0082]when said plasmid comprises a sequence encoding a Met28 protein,
then said marker is chosen from antigenic peptides, intrinsic
fluorescence proteins and proteins with measurable enzymatic activity,
and

[0083]when said plasmid comprises a sequence encoding a Met30 protein,
then said marker is chosen from intrinsic fluorescence proteins and
proteins with measurable enzymatic activity.

[0084]According to an advantageous embodiment of said plasmids, they are
selected from the group consisting of:

[0085]plasmids containing a sequence encoding a Met4 or Met28 protein
fused with a peptide comprising three hemagglutinin (HA) antigenic units,
said sequence being expressed under the control of the GAL1 inducible
promoter,

[0086]plasmids containing a sequence encoding a Met4, Met28 or Met30
protein, fused with a GFP protein, said sequence being respectively
expressed under the control of the constitutive promoters MET4, MET28 or
MET30 or under the control of the GAL1 inducible promoter,

[0087]plasmids containing a sequence encoding a Met30 protein, fused with
a GFP protein and with a peptide comprising 3 hemagglutinin (HA)
antigenic units, said sequence being expressed under the control of the
GAL1 promoter.

[0088]The subject of the present invention is also plasmids, characterized
in that they contain a hybrid sequence comprising a sequence encoding a
Met4 protein, in its wild-type or mutated form, fused in phase with at
least one sequence encoding the DNA-binding factor LexA, and the TRP1
gene or the LEU2 gene as genes for selecting yeasts modified with said
plasmids.

[0089]The subject of the present invention is also plasmids containing a
reporter transcriptional system consisting of a reporter gene placed
under the control either of an appropriate yeast promoter or of an
appropriate operator sequence, as defined above.

[0090]Advantageously, said plasmids contain the LacZ or XylE reporter
gene, expressed under the control, either of the MET16 promoter, or of
LexA operators.

[0091]The subject of the present invention is also yeast strains,
characterized in that they are stably modified with at least one plasmid
according to the present invention.

[0092]Other characteristics and advantages of the invention appear in the
remainder of the description and the examples, which are illustrated by
figures in which:

[0093]FIG. 1 illustrates the influence of the addition of methionine at
repressive concentrations to the culture medium (A) the cells containing
a plasmid coding the proteins labeled with hemagglutinin (Ha) antigenic
units under the control of the GAL1 promoter, and prepared according to
the procedure described in examples 2 and 4, are cultured, according to
the procedure described in example 12, in a minimum medium containing 2%
galactose for 90 minutes, or in the presence of methionine at a
repressive concentration (+Met), or in the absence of methionine (-Met);
(B) and (C) the total RNAs are extracted, according to the procedure
described in example 12 from cells used in (A) and expressing either the
Ha-Met4 hybrid protein, or the Ha-Met28 hybrid protein. The cells are
cultured under the conditions described in (A) and are analyzed with
MET4, MET16, MET25, MET28. "met4Δ" corresponds to a W303 cell
modified with a chromosomal copy of the inactivated MET4 gene;
"met28Δ" corresponds to a W303 cell modified with a chromosomal
copy of the inactivated MET28 gene.

[0094]FIG. 2 illustrates the location of the GFP-Met4 and GFP-Met28 hybrid
proteins in wild-type cells, in the absence of methionine (-Met) or in
the presence of methionine at a repressive concentration (+Met). The
cells are cultured under the conditions described in example 12. "Hoe"
corresponds to the colored indicator specific to the nuclei, Hoechst
333-42; "Nom" corresponds to the image obtained by Nomarski interference
microscopy.

[0095]FIG. 3 illustrates (A) the location of the GFP-Met30 hybrid protein
in wild-type cells, in the absence of methionine (-Met) or in the
presence of methionine at a repressive concentration (+Met). The cells
are cultured under the conditions described in example 12. "Hoe"
corresponds to the colored indicator Hoechst 333-42; "Nom" corresponds to
the image obtained by Nomarski interference microscopy; (B) stability of
the Ha-Met30 and Ha-Met30ΔF hybrid proteins in wild-type W303-1A
strains.

[0096]FIG. 4 illustrates the activity of the LexAopXylE reporter gene in
the absence of methionine (-Met) or in the presence of methionine at a
repressive concentration, from a yeast strain (C190) expressing the
hybrid protein encoded by the plasmid pLexMet4-7; the visualization is
carried out by measuring catechol oxidase.

EXAMPLE 1

Construction of the Basic Plasmids pGal316Flu, pGal306Flu and pFL39Flu

[0100]The fragment obtained is digested with the restriction enzymes
HindIII and EcoRI and inserted:

[0101](i) into the S. cerevisiae-E. coli shuttle plasmid pRS306 whose
sequence has been deposited in the EMBL databank, with the identifier
"PRS316", under the No. U03442, digested beforehand with the enzymes
HindIII and EcoRI, producing the plasmid pGal316.

[0102](ii) into the S. cerevisiae-E. coli shuttle plasmid pRS306 whose
sequence has been deposited in the EMBL databank, with the identifier
"PRS306", under the No. U03438, digested beforehand with the restriction
enzymes HindIII and EcoRI.

[0106](i) into the plasmid pGal316 obtained at point 1.1.1., digested
beforehand with the restriction enzyme EcoRI and whose ends have been
made blunt by treatment with the Klenow fragment of E. coli DNA
polymerase, producing the plasmid pGal316Flu,

[0107](ii) into the plasmid pGal306 prepared at point 1.1.1., digested
beforehand with the restriction enzyme EcoRI and whose ends have been
made blunt by treatment with the Klenow fragment of E. coli DNA
polymerase, producing the plasmid pGal306Flu.

[0108]1.2. Construction of the Plasmid pFL39Flu:

[0109]In a first stage, the EcoRI site present in the polylinker of the S.
cerevisiae-E. coli shuttle plasmid pFL39 was destroyed. For that, the
plasmid pFL39, whose sequence is that deposited in the EMBL databank,
with the identifier "CVPFL39", under the No. X70483, was digested with
EcoRI, the ends made blunt by treating with the Klenow fragment of E.
coli DNA polymerase, and the product thus treated was self-ligated,
producing the plasmid pFL39E0.

[0110]In a second stage, the HindIII-PstI fragment of the plasmid
pGal316Flu prepared according to the procedure described at point 1.1.
and comprising the GAL1 promoter region and the region encoding the Ha
antigenic region was inserted into the plasmid pFL39E0 digested
beforehand with the HindIII and PstI enzymes.

[0111]The plasmid pFL39Flu is thus obtained.

EXAMPLE 2

Plasmids Allowing the Expression in Yeast of Ha-Met4 Hybrid Proteins Under
the Control of the GAL1 Promoter.

[0112]The plasmids which follow allow the expression, in the S. cerevisiae
yeast, of derivatives of the Met4 protein comprising a repetition of
three hemagglutinin (Ha) antigenic units at their amino-terminal end.

[0129]This plasmid allows the expression, under the control of the MET4
promoter, of a GFP-Met4 hybrid protein comprising amino acids 15 to 666
of Met4, and can replicate autonomously in yeast.

[0130]3.2. Construction of the Plasmid pGal316GFPMet4

[0131]The EcoRI-BamHI fragment of the plasmid pGFPMet4, encoding the
GFP-Met4 fusion, was inserted into the plasmid pGal316 digested
beforehand with the enzymes EcoRI and BamHI, producing the plasmid
pGal316GFPMet4. This plasmid can replicate autonomously in yeast.

[0132]3.3. Construction of the Plasmid pGal316GFPMet4Δ12:

[0133]The EcoRI-EcoRI DNA fragment of the vector pGal316GFPMet4 prepared
according to the procedure described above and encoding GFP was cloned in
phase into the plasmid pGal316FluMet412 digested beforehand with the
enzyme EcoRI, producing the plasmid pGal316GFPMet4Δ12. This plasmid
can replicate autonomously in yeast.

[0134]3.4. Construction of the Plasmid pGal316GFPMet4Δ30:

[0135]The EcoRI-EcoRI DNA fragment of the vector pGal316GFPMet4 prepared
according to the procedure described at point 3.2 and encoding GFP was
cloned in phase into the plasmid pGal316FluMet4Δ30 digested
beforehand with the enzyme EcoRI, producing the plasmid
pGal316GFPMet4Δ30. This plasmid can replicate autonomously in
yeast.

[0136]3.5. Construction of the Plasmid pGal316GFPMet4Δ37:

[0137]The EcoRI-EcoRI DNA fragment of the vector pGal316GFPMet4 prepared
according to the procedure described at point 3.2 and encoding GFP was
cloned in phase into the plasmid pGal316FluMet4Δ37 digested
beforehand with the enzyme EcoRI, producing the plasmid
pGal316GFPMet4Δ37. This plasmid can replicate autonomously in
yeast.

[0138]3.6. Construction of the Plasmid pGal306GFPMet4:

[0139]The Not1-Asp718 DNA fragment of the vector pGal316GFPMet4 prepared
according to the procedure described at point 3.2 and comprising the
entire GAL1 promoter contained in the sequence deposited at the EMBL
databank under the identifier "SCGAL10", under the No. K02115, and the
GFP-Met4 fusion (residues 15 to 666 of Met4) was inserted into the
plasmid pRS306 digested beforehand with the Not1-Asp718 enzymes,
producing the plasmid pGal306GFPMet4.

[0140]3.7. Construction of the Plasmid pGal306GFPMet4Δ12:

[0141]The Not1-Asp718 DNA fragment of the vector pGal316GFPMet4Δ12
prepared according to the procedure described at point 3.3 and comprising
the entire GAL1 promoter and the GFP-Met4Δ12 fusion (residues 1579
and 180-666 of Met4) was inserted into the plasmid pRS306 digested
beforehand with the Not1-Asp718 enzymes, producing the plasmid
pGal306GFPMet4Δ12.

[0142]3.8. Construction of the Plasmid pGal306GFPMet4Δ30:

[0143]The Not1-Asp718 DNA fragment of the vector pGal316GFPMet4Δ30
prepared according to the procedure described at point 3.4 and comprising
the entire GAL1 promoter and the GFP-Met4Δ30 fusion (residues 15 to
211 and 221 to 666 of Met4) was inserted into the plasmid pRS306 digested
beforehand with the Not1-Asp718 enzymes, producing the plasmid
pGal306GFPMet4Δ30.

[0144]3.9. Construction of the Plasmid pGal306GFPMet4Δ37:

[0145]The Not1-Asp718 DNA fragment of the vector pGal316GFPMet4Δ37
prepared according to the procedure described at point 3.5 and comprising
the entire GAL1 promoter and the GFP-Met4Δ37 fusion (residues 15 to
352 and 366 to 666 of Met4) was inserted into the plasmid pRS306 digested
beforehand with the Not1-Asp718 enzymes, producing the plasmid
pGal306GFPMet4Δ37.

EXAMPLE 4

Construction of the Plasmids Allowing the Expression in Yeast of Ha-Met28
and GFP-Met28 Hybrid Proteins

[0146]4.1. Construction of the Plasmid pGal316FluMet28:

[0147]The EcoRI-BamHI DNA fragment of the vector pLexM28-2 (Kuras L. et
al., (1996), EMBO J., 15, 2519-2529) encoding amino acids 1 to 166 of the
Met28 protein was cloned into the plasmid pGal316Flu digested beforehand
with the enzymes EcoRI and BamHI, producing the plasmid pGal316FluMet28.
This plasmid can replicate autonomously in yeast. It allows the
expression in yeast of a full-length Met28 protein comprising, at its
amino-terminal end, a repetition of 3 hemagglutinin (Ha) antigenic units.
The Ha-Met28 hybrid protein is expressed under the control of the GAL1
promoter.

[0148]4.2. Construction of the Plasmid pDFL39FluMet28:

[0149]The EcoRI-BglII DNA fragment of the vector pLexM28-2 (Kuras L. et
al., (1996), reference cited) encoding amino acids 1 to 166 of the Met28
protein was cloned into the plasmid pFL39Flu digested beforehand with the
enzymes EcoRT and BglII, producing the plasmid pFL39FluMet28. This
plasmid can replicate autonomously in yeast. It allows the expression in
yeast of a full-length Met28 protein comprising, at its amino-terminal
end, a repetition of 3 hemagglutinin (Ha) antigenic units. The Ha-Met28
hybrid protein is expressed under the control of the GAL1 promoter.

[0150]4.3. Construction of the Plasmid pGFPMet28:

[0151]4.3.1. Construction of the plasmid p314Met28:

[0152]The XbaI-HpaI DNA fragment of the vector pMet28-1 (Kuras L. et al.,
(1996), reference cited) containing the MET28 gene, whose sequence is
that deposited at the EMBL databank, under the identifier "SC17015",
under the No. U17015 and its promoter region was isolated, its ends made
blunt by treating with the Klenow fragment of E. coli DNA polymerase and
this DNA fragment was inserted into the plasmid pRS314 (Sikorski R. S. et
al., (1989), Genetics, 122, 19-27) digested beforehand with the enzyme
SmaI, producing the plasmid p314Met28. This plasmid can replicate
autonomously in yeast.

[0153]4.3.2. Construction of the Plasmid p314GFPMet28:

[0154]A fragment of 710 base pairs (bp) of the plasmid pGFPmut3, encoding
the GFP3 protein, was amplified by PCR using the oligonucleotides
"olM28GFP5" having the sequence SEQ ID No. 6:

[0156]The fragment obtained was inserted into the plasmid p314Met28 by the
"gap repair" method according to the technique described by Orr-Weaver et
al. (Methods in Enzymology, (1983), 101, 228-245).

[0157]Thus, the fragment obtained after PCR and the plasmid p314Met28,
digested beforehand with the enzyme BglII, were cotransformed into the
yeast strain W303-1A (Bailis A. M. et al., (1990), reference cited), and
the clones prototrophic for tryptophan were selected. The plasmid DNA
contained in these clones was extracted, transformed into E. coli and the
recombinant plasmid p314Met28GFP identified and characterized by
enzymatic digestions and sequence. This plasmid allows the expression of
a full-length Met28 protein fused at its carboxyl-terminal end with the
GFP protein. The Met28-GFP hybrid protein is expressed under the control
of the MET28 promoter. This plasmid can replicate autonomously in yeast.

EXAMPLE 5

Construction of the Plasmids Allowing the Expression in Yeast of Ha-Met30
and GFP-Met30 Hybrid Proteins

[0158]5.1. Construction of the Plasmid pGal316FluMet30ΔN:

[0159]The EcoRI-BglII DNA fragment of the vector pDGadMet30-1 (Thomas D.
et al. reference cited) encoding amino acids 158 to 640 of the Met30
protein was cloned into the plasmid pGal316Flu digested beforehand with
the enzymes EcoRI and BamHI, producing the plasmid
pGal316FluMet30ΔN. This plasmid can replicate autonomously in
yeast. It allows the expression in yeast of a Met30 protein truncated of
its amino-terminal portion but comprising at this end a repetition of 3
hemagglutinin (Ha) antigenic units. The Ha-Met30 hybrid protein is
expressed under the control of the GAL1 promoter.

[0160]5.2. Construction of the Plasmid pGal316FluMet30:

[0161]The EcoRI-EcoRI DNA fragment of the vector pLexMet30-4 (Thomas D. et
al., (1995), reference cited) encoding the first 157 amino acids of the
Met30 protein was cloned into the plasmid pGal316FluMet30ΔN
digested beforehand with the enzyme EcoRI, producing the plasmid
pGal316FluMet30. This plasmid can replicate autonomously in yeast. It
allows the expression in yeast of a full-length Met30 protein comprising,
at its amino-terminal end, a repetition of 3 hemagglutinin (Ha) antigenic
units. The Ha-Met30 hybrid protein is expressed under the control of the
GAL1 promoter.

[0162]5.3. Construction of the Plasmid pFL39FluMet30ΔN:

[0163]The EcoRI-BglII DNA fragment of the vector pGadMet30-1 (Thomas D. et
al., (1995), reference cited) encoding amino acids 158 to 640 of the
Met30 protein was cloned into the plasmid pFL39Flu digested beforehand
with the enzymes EcoRI and BamHI, producing the plasmid
pFL39FluMet30ΔN. This plasmid can replicate autonomously in yeast.
It allows the expression in yeast of a protein of a protein Met30
truncated of its amino-terminal portion but comprising at this end a
repetition of 3 hemagglutinin (Ha) antigenic units. The Ha-Met30 hybrid
protein is expressed under the control of the GAL1 promoter.

[0164]5.4. Construction of the Plasmid pFL39FluMet30ΔF:

[0165]This plasmid is constructed according to the procedure described at
point 5.3, but expresses a mutated Met30 protein which does not comprise
amino acids 187 to 201.

[0166]5.5. Construction of the Plasmid pFL39FluMet30

[0167]The EcoRI-EcoRI DNA fragment of the vector pLexMet30-4 (Thomas D. et
al., (1995), reference cited) encoding the first 157 amino acids of the
Met30 protein was cloned into the plasmid pFL39FluMet30ΔN digested
beforehand with the enzyme EcoRI, producing the plasmid pFL39FluMet30.
This plasmid can replicate autonomously in yeast. It allows the
expression in yeast of a full-length Met30 protein comprising, at its
amino-terminal end, a repetition of 3 hemagglutinin (Ha) antigenic units.
The Ha-Met30 hybrid protein is expressed under the control of the GAL1
promoter.

[0168]5.6. Construction of the Plasmid p316FluMet30GFP:

[0169]A fragment of 710 base pairs (bp) of the plasmid pGFPmut3, encoding
the GFP3 protein, was amplified by PCR using the oligonucleotides
"olM30GFP5" having the sequence SEQ ID No. 8:

[0172]Thus, the fragment obtained after PCR and the plasmid
pGal316FluMet30, digested beforehand with the enzyme XbaI, were
cotransformed into the yeast strain W303-1A and the clones prototrophic
for uracil were selected. The plasmid DNA contained in these clones was
extracted, transformed into E. coli and the recombinant plasmid
p316FluMet30GFP identified and characterized by enzymatic digestions and
sequence. This plasmid allows the expression of a full-length Met30
protein, fused at its carboxyl-terminal end with the GFP protein and at
its amino-terminal end with a repetition of 3 hemagglutinin (Ha)
antigenic units. The Ha-Met30-GFP hybrid protein is expressed under the
control of the GAL1 promoter. This plasmid can replicate autonomously in
yeast.

[0173]5.7. Construction of the Plasmid pMet30GFP:

[0174]5.7.1. Construction of the plasmid pMet30-8:

[0175]The XbaI-SalI DNA fragment of the vector pMet30-1 (Thomas D. et al.,
(1995), reference cited) containing the MET30 gene whose sequence is that
deposited at the EMBL databank, under the identifier "SCMET30A", under
the No. L26505 and its promoter region was isolated and inserted into the
plasmid pRS316 digested beforehand with the enzymes XbaI and SalI,
producing the plasmid pMet30-8. This plasmid can replicate autonomously
in yeast.

[0176]5.7.2. Construction of the Plasmid pMet30GFP:

[0177]A fragment of 710 base pairs (bp) of the plasmid pGFPmut3, encoding
the GFP3 protein, was amplified by PCR using the oligonucleotides
"olM30GFP5" having the sequence SEQ ID No. 10:

[0179]Thus, the fragment obtained after PCR and the plasmid pMet30-8,
digested beforehand with the enzyme NheI, were cotransformed into the
yeast strain W303-1A and the clones prototrophic for uracil were
selected.

[0180]The plasmid DNA contained in these clones was extracted, transformed
into E. coli and the recombinant plasmid pMet30GFP identified and
characterized by enzymatic digestions and sequence. This plasmid allows
the expression of a full-length Met30 protein, fused at its
carboxyl-terminal end, with the GFP protein. The Met30-GFP hybrid protein
is expressed under the control of the MET30 promoter. This plasmid can
replicate autonomously in yeast.

EXAMPLE 6

Construction of the Plasmids pYi39XyLex, pYi44XyLex, pLexM4-7,
pLexM4Δ239, pM16Z1 and pM16Xyl1 which Make it Possible to Visualize
the Transcriptional Activity of the Yeast MET Genes

[0181]6.1. Construction of the Plasmid pYi39XyLex:

[0182]The integration of this plasmid into the yeast genome at the TRP1
locus can be directed by linearizing this plasmid with the enzyme Stu1
and by transforming, with the plasmid thus linearized, a yeast strain
carrying a point mutation in the trp1 gene.

[0183]6.1.1. Construction of the Plasmid pXylex:

[0184]The DNA fragment containing the promoter of the GAL1 gene whose
transcription activating sequences have been replaced by E. coli LexA
protein binding sites, whose sequence is that deposited at the EMBL bank,
under the identifier "ECLEXA1", under the No. J01643, was amplified by
PCR from the plasmid pSH18-34 (Hanes S. D. et al., (1989), Cell, 57,
1275-1293) using the oligonucleotides "olGAL1" having the sequence SEQ ID
No. 12 5'GCCAAGCTTCTCCTTGACGTTAAAGTA3' and "olGAL10" having the sequence
SEQ ID No. 13 5'GCCGGATCCTTTGTAACTGAGCTGTCA3'. The amplified DNA was
digested with the restriction enzymes BamHI and HindIII and inserted into
the vector pEMBLYe31-X (Jacquemin-Faure I. et al., (1994), Mol. Gen.
Genet., 244, 519-529) digested beforehand with the enzymes BamHI and
HindIII, producing the vector pXylex.

[0185]6.1.2. Construction of the Plasmid pYi39:

[0186]The vector pFL39 was digested with the enzyme ClaI and was
self-ligated and a plasmid having lost the ClaI-ClaI fragment containing
the original ars-cen replication origin was selected, producing the
vector pYi39.

[0187]6.1.3. Construction of the Plasmid pYi39Xylex:

[0188]The BamHI-PstI DNA fragment of the vector pXylex prepared beforehand
and containing the XylE gene whose sequence is that deposited at the EMEL
bank, under the identifier "PPXYLE", under the No. V01161 preceded by the
GAL1-LexA promoter was isolated and inserted into the plasmid pYi39
digested beforehand with the enzymes BamHI and PstI, producing the
plasmid pYi39Xyl-Lex.

[0189]6.2. Construction of the Plasmid pYi44Xylex:

[0190]6.2.1. Construction of the Plasmid pYi44:

[0191]The vector pFL44 was digested with the enzyme ClaI and was
self-ligated and a plasmid having lost the ClaI-ClaI fragment containing
the original 2 μm replication origin was selected, producing the
vector pYi44.

[0192]6.2.2. Construction of the Plasmid pYi44Xylex

[0193]The BamHI-PstI DNA fragment of the vector pXylex prepared according
to the procedure described at point 6.1.1 and containing the XylE gene
preceded by the GAL1-LexA promoter region was isolated and inserted into
the plasmid pYi44 digested beforehand with the enzymes BamHI and PstI,
producing the plasmid pYi44Xyl-Lex.

[0194]6.3. Construction of the Plasmid pLexM4-7:

[0195]6.3.1. Use:

[0196]It allows the expression in the yeast S. cerevisiae of the Met4
protein containing at its amino-terminal end the 202 residues of the E.
coli LexA protein. The hybrid protein thus synthesized is capable of
binding to DNA regions comprising the LexA operators.

[0201]It allows the expression in the yeast Saccharomyces cerevisiae of a
derivative of the Met4 protein from which there have been amputated its
residues 212 to 231 comprising at its amino terminal end the 202 residues
of the E. coli LexA protein. The hybrid protein thus synthesized is
capable of binding to DNA regions comprising LexA operators.

[0202]6.4.2. Construction:

[0203]The EcoRI-BmHI DNA fragment of the vector pLexM4Δ30 encoding a
derivative comprising amino acids 15 to 211 and 232 to 666 of the Met4
protein was cloned into the plasmid pBTM116 (Margottin M. et al., (1998),
reference cited) digested beforehand with the enzymes EcoRI and BamHI,
producing the plasmid pLexM4Δ239. This plasmid can replicate
autonomously in yeast.

[0204]6.5. Construction of the Plasmid pM16Z1:

[0205]The DNA fragment containing the promoter of the MET16 gene
(comprising nucleotides -535 to +3, numbered from the initiation codon of
the MET16 gene) was amplified by PCR from the plasmid pM16-1 (Hanes S. D.
et al., (1989), reference cited) using the oligonucleotides "M16OL2"
having the sequence SEQ ID No. 14 5'CAACGAAGGATCCAATAATCGAAGCC3' and
"M16OL4" having the sequence SEQ ID No. 15
5'GGGGAATTCCTTCATTTTATGAGTTGCT3'. The amplified DNA was digested with the
restriction enzymes BamHI and EcoRI and inserted into the vector Yep356R
(Myers A. M. et al. (1986), Gene, 45, 299-310) digested beforehand with
the enzymes BamHI and EcoRII, producing the vector pM16Z1. This plasmid
can replicate autonomously in yeast and allows the expression of E. coli
β-galactosidase under the control of the MET16 promoter.

[0206]6.6. Construction of the Plasmid pM16Xyl1:

[0207]The DNA fragment containing the promoter of the MET16 gene
(comprising nucleotides -535 to -1, numbered from the initiation codon of
the MET16 gene) was amplified by PCR from the plasmid pM16-1 (Thomas D.
et al. (1990), J. Biol. Chem., 265, 15518-15524) using the
oligonucleotides "M16OL5" having the sequence SEQ ID No. 16
5'CAACGAAGCTTTCAATAATCGAAGCACTTGG3' and "M16OL6" having the sequence SEQ
ID No. 17 5'TTTATGAGAAGCTTTGGGTTGATACCTTTGC3'. The amplified DNA was
digested with the restriction enzyme HindIII and inserted into the vector
pUC9-LEU2-X (Jacquemin-Faure I. et al., (1994), reference cited)
producing the plasmid pM16Xyl1. The integration of this plasmid into the
yeast genome at the LEU2 locus can be directed by linearizing this
plasmid with the enzyme Asp718 and by transforming, with the plasmid thus
linearized, a yeast strain carrying a point mutation in the leu2 gene.
Thus, this plasmid makes it possible to obtain a modified yeast strain
stably expressing P. putida catechol oxidase placed under the control of
the MET16 gene.

EXAMPLE 7

Yeast Strains Expressing a GFP-Met4 Hybrid Protein

[0208]These strains carry at the level of the URA3 locus (chromosome V,
left arm) an artificial gene allowing the expression of a Met4 protein,
modified or otherwise, under the control of the promoter of the GAL1
gene.

[0209]The correct integration into the URA3 locus of the GAL-Met4-GFP
fusions was verified by conventional molecular biology and genetic
techniques.

[0212]The integration of this plasmid into the yeast genome at the URA3
locus can be directed by linearizing this plasmid with the enzyme Stu1
and by transforming, using the plasmid thus linearized, a yeast strain
carrying a point mutation in the ura3 gene. The GFP-Met4 fusion thus
integrated into the URA3 chromosomic locus is expressed under the control
of the GAL1 promoter.

[0213]7.2. Preparation of the Strains from pGal306GFPMet4Δ12:

[0214]The strains are transformed according to the procedure described at
point 7.1. from the plasmid pGal306GFPMet4Δ12.

[0215]7.3. Preparation of the Strains from pGal306GFPMet4Δ30:

[0216]The strains are transformed according to the procedure described at
point 7.1. from the plasmid pGal306GFPMet4Δ30.

[0217]7.4. Preparation of the Strains from pGal306GFPMet4Δ37:

[0218]The strains are transformed according to the procedure described at
point 7.1. from the plasmid pGal306GFPMet4Δ37.

EXAMPLE 8

Yeast Strains Carrying the XyLE Gene Encoding Catechol Oxidase Under the
Control of LexA Operators

[0219]8.1. Preparation of the Strains:

[0220]These strains carry at the level of the URA3 locus (chromosome V,
left arm) or at the level of the TRP1 locus (chromosome IV, right arm) an
artificial gene comprising the Pseudomonas putida XylE gene placed under
the control of LexA operators. The expression of this gene is directed by
the LexA-Met4 hybrid protein expressed from the replicative plasmid
pLexM4-7.

[0221]The correct integration into the URA3 locus of the GAL-Met4-GFP
fusions was checked by conventional molecular biology and genetic
techniques.

[0222]The strains which were prepared are assembled in table II below.

[0224]The integration of this plasmid into the yeast genome at the URA3
locus can be directed by linearizing this plasmid with the Stu1 enzyme
and by transforming, with the plasmid thus linearized, a yeast strain
carrying a point mutation in the ura3 gene (Orr-Weaver T. L. et al.
(1983) and Rothstein R. (1991), references cited).

[0225]8.3. Preparation of the Strains from the Plasmid pYi39XyLex

[0226]The integration of this plasmid into the yeast genome at the TRP1
locus can be directed by linearizing this plasmid with the Stu1 enzyme
and by transforming, with the plasmid thus linearized, a yeast strain
carrying a point mutation in the trp1 gene (Orr-Weaver T. L. et al.
(1983) and Rothstein R. (1991), references cited).

EXAMPLE 9

Yeast Strain Carrying the XylE Gene Encoding Catechol Oxidase Under the
Control of the Promoter of the MET25 Gene

[0227]9.1. Preparation of the CC634-2D Strain:

[0228]This strain is derived from the cross between the CI2-11D strain
(Jacquemin-Faure I. et al., (1994), reference cited) and the CD106 strain
(Thomas D. et al., (1992), reference cited). It contains, integrated at
the LEU2 locus (chromosome III, left arm), an XylE gene placed under the
control of the promoter of the MET25 gene.

Yeast Strain Carrying the HIS3 Gene Under the Control of LexA Operators

[0231]These strains carry at the level of the LYS2 locus (chromosome II,
right arm) an artificial gene comprising the S. cerevisiae HIS3 gene
placed under the control of LexA operators. The expression of this gene
is directed by the LexA-Met4 hybrid protein expressed from the
replicative plasmid pLexM4-7.

Yeast Strain Carrying the LacZ Gene Under the Control of LexA Operators

[0242]These strains carry at the level of the URA3 locus (chromosome V,
left arm) an artificial gene comprising the E. coli LacZ gene placed
under the control of LexA operators. The expression of this gene is
directed by the LexA-Met4 hybrid protein expressed from the replicative
plasmid pLexM4-7.

Determination of the Level of Expression and of the Stability of the
Proteins Expressed from the Hybrid Sequences Contained in the Plasmids
According to the Invention

[0247]12.1. Procedure:

[0248]12.1.1. Determination of the Stability of the Hybrid Proteins:

[0249]a) Visualization with the Hemagglutinin (Ha) Antigenic Marker:

[0250]Yeast cells containing a plasmid encoding the proteins labeled with
Ha, under the control of the GAL1 promoter, are cultured in a medium
containing raffinose.

[0251]The induction of the hybrid protein, expressed under the control of
the GAL1 promoter, is carried out for 2 to 10 hours, by transferring the
cells into a medium containing 2 to 5% galactose.

[0252]Samples were collected at 0, 5, 10, 20, 40, 60 and 80 minutes after
the addition of glucose and optionally methionine, at a repressive
concentration of between 0.05 mM and 25 mM.

[0253]The stability of the hybrid proteins is measured by reaction with
anti-Ha antibodies, according to conventional techniques.

[0254]b) Visualization with the GFP Protein:

[0255]Yeast cells containing a plasmid encoding the proteins labeled with
the GFP protein, under the control of the GAL1 promoter, are cultured in
a medium containing raffinose and the induction of the hybrid protein is
carried out according to the procedure described above.

[0256]The measurement of fluorescence is carried out after 20 minutes of
incubation in the presence or in the absence of repressive concentrations
of methionine.

[0257]c) Visualization with Catechol Oxidase:

[0258]The activity of the reporter gene LexAopXyle is measured in a yeast
strain (C190) expressing the hybrid protein encoded by the plasmid
pLexMet4-7 and cultured under the conditions described above.

[0259]The activity is measured by a visualization technique based on
measuring catechol oxidase according to conventional techniques.

[0260]12.1.2. Measurement of the Total RNAs:

[0261]It is carried out by conventional techniques known to a person
skilled in the art.

[0262]12.2. Results:

[0263]12.2.1. Stability of the Hybrid Proteins:

[0264]a) Visualization by the Hemagglutinin (Ha) Antigenic Marker

[0265]They are assembled in FIGS. 1 and 3B.

[0266]Expression of the Ha-Met4 hybrid protein

[0267]The Ha-Met4 protein has a half-life of the order of 20 minutes in
the absence of methionine (FIG. 1A, -Met) and a half-life of the order of
5 minutes under repressive conditions, that is to say in the presence of
methionine (FIG. 1A, +Met).

[0268]The degradation of the Ha-Met4 protein is almost complete under
repressive conditions after 20 minutes (FIG. 1A, +Met)

[0269]Expression of the Ha-Met28 hybrid protein

[0270]The Ha-Met28 protein has a half-life of the order of 20 minutes in
the absence of methionine (FIG. 1A, -Met).

[0271]Repressive conditions do not modify the half-life of the Ha-Met28
protein (FIG. 1A, +Met).

[0272]Expression of the Ha-Met30 and Ha-Met30ΔF hybrid proteins:

[0273]The Ha-Met30 protein has a half-life of the order of 20 minutes in
the absence of methionine (FIG. 3B, -Met).

[0274]Repressive conditions do not modify the half-life of the Ha-Met30
hybrid protein (FIG. 3B, +Met).

[0275]The Ha-Met30ΔF hybrid protein appears less stable than the
Ha-Met30 hybrid protein, but repressive conditions do not reduce the
stability of this hybrid protein.

[0276]b) Visualization with Hybrid Proteins Comprising GFP

[0277]The results are illustrated in FIGS. 2 and 3B.

[0278]Location of the GFP-Met4 hybrid protein

[0279]The GFP-Met4 hybrid protein is located in the nucleus when the cells
are cultured in the absence of methionine.

[0280]On the other hand, under repressive conditions, that is to say in
the presence of methionine, this location is no longer observed (FIG.
2A).

[0281]These results are in agreement with a rapid degradation of the
hybrid protein under repressive conditions.

[0282]Location of the GFP-Met28 hybrid protein

[0283]The GFP-Met28 hybrid protein is always present in the nucleus,
whether in the absence or in the presence of methionine (FIG. 2B).

[0284]Location of the GFP-Met30 hybrid protein

[0285]The GFP-Met30 hybrid protein is always present in the nucleus,
whether in the absence or in the presence of methionine (FIG. 3A).

[0286]12.2.2. Expression of Total RNAs

[0287]The results are illustrated in FIGS. 1B and 1C

[0288]The levels of Met4 and of Met28 allow the activation of the
transcription of the target genes.

[0289]Neither the expression of MET4, nor that of MET28, under the control
of the GAL1 promoter, modifies the repression induced by the addition of
methionine.

[0290]12.2.3. Visualization with Catechol Oxidase

[0291]The results are illustrated in FIG. 4.

[0292]In the absence of methionine, the cells are stained yellow, proof
that the protein is expressed.

[0293]In the presence of methionine, the cells are white because of the
repression.